CN113620963A

CN113620963A - Mitochondrial viscosity probe and preparation method and application thereof

Info

Publication number: CN113620963A
Application number: CN202111028020.6A
Authority: CN
Inventors: 张洪星; 刘景�; 郭炜
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2021-09-02
Filing date: 2021-09-02
Publication date: 2021-11-09
Anticipated expiration: 2041-09-02
Also published as: CN113620963B

Abstract

The invention relates to the field of fluorescent probes, in particular to a mitochondrial viscosity probe and a preparation method and application thereof. In order to solve the problem that most viscosity probes lack the mitochondrion targeting capability, the probe is prepared by coupling a carbon dipyrromethane dye (Cardipy) and a benzene ring through chemical reaction. The probe has only weak fluorescence signal due to the nonradiative transition process caused by the rotation of the benzene ring; as the viscosity increases, the nonradiative transition process caused by the rotation of the benzene ring is effectively suppressed and the probe exhibits a strong fluorescent signal. In addition, the probe has good water solubility, high chemical and light stability and excellent mitochondrial targeting capability, and is successfully used for detecting the viscosity change in cell mitochondria before and after drug induction.

Description

Mitochondrial viscosity probe and preparation method and application thereof

Technical Field

The invention relates to the field of fluorescent probes, in particular to a mitochondrial viscosity probe and a preparation method and application thereof.

Background

The microenvironment of cells mainly comprises viscosity, polarity, temperature, hypoxia, acidity and alkalinity and the like, and the stability of the microenvironment is an important condition for keeping normal proliferation, differentiation, metabolism and functional activities of cells. Wherein, the cell viscosity is an important parameter for reflecting the flowing state of substances such as protein, lipid, polysaccharide and the like, has important significance for signal transmission and interaction between biomolecules in cells, and influences the execution of cell functions such as apoptosis and autophagy; abnormal cell viscosity is closely related to various diseases such as alzheimer's disease, diabetes and cancer. Mitochondria is one of the subcellular organelles of most interest in the study of fluorescence bioimaging, and the organelle is the main site of cellular respiration and is involved in the processes of cell differentiation, information transmission, apoptosis, growth regulation and the like besides supplying energy to cells. The viscosity of mitochondria plays an important role in regulating the biological process, reflecting the condition and function of organelles; abnormal mitochondrial viscosity can lead to cellular disorders or serious diseases, for example, an increase in mitochondrial viscosity decreases the activity of the electron transport chain, promotes the release of cytochrome C, and ultimately increases the incidence of malignancy and atherosclerosis. It has long been speculated that abnormal mitochondrial viscosity is associated with neurodegenerative diseases, atherosclerosis and diabetes. Therefore, real-time in situ monitoring of changes in cell viscosity is of great significance for understanding the manifestation of cell function and elucidating the mechanism of progression of the associated disease.

Conventional viscometers (e.g., capillary, rotational, and ball viscometer) are only suitable for detecting the viscosity of a fluid, and are not suitable for detecting organisms. Fluorescence technology has become one of the most powerful tools in cell biology for studying biological phenomena due to its inherent advantages of visualization, non-invasiveness, sensitivity, and real-time monitoring. With this technique, imaging and tracking of various biological species in living cells or whole organisms can be achieved with high spatial and temporal resolution, which greatly improves our understanding of biological systems and also facilitates drug development, clinical diagnosis and disease treatment. At present, a large number of viscosity fluorescent probes are reported, whose chemical structure is usually composed of a fluorophore bound to a group that can rotate with respect to the whole molecule. Under low viscosity conditions, the fast rotation consumes the excited state energy of the molecule, and the probe transitions back to the ground state in a non-radiative form, resulting in low fluorescence quantum yield and short fluorescence lifetime; with the increase in viscosity, the rotation is effectively inhibited, the probability of non-radiative transitions is reduced, and the fluorescence of the probe is restored. However, most viscosity probes reported so far show non-specific distribution in cells due to lack of mitochondrial targeting ability, and additional targeting groups (such as triphenylphosphine salt and quaternary ammonium salt) have to be introduced into their structures in order to localize such probes to mitochondria. In the invention, a novel viscosity probe is developed, and the probe has the advantages of good water solubility, strong chemical and light stability and the like; importantly, due to the characteristics of positive ions, the probe has excellent water solubility, cell membrane penetrability and natural mitochondrial targeting. The probe is successfully used for detecting the viscosity change of mitochondria in cells under the induction of drugs.

Disclosure of Invention

Aiming at the problems, the invention provides a viscosity fluorescent probe with mitochondrion targeting capability and preparation and application thereof, wherein the probe is prepared by coupling a carbon dipyrromethene dye (Cardipy) and a benzene ring through chemical reaction. The probe has only weak fluorescence signal due to the nonradiative transition process caused by the rotation of the benzene ring; as the viscosity increases, the nonradiative transition process caused by the rotation of the benzene ring is effectively suppressed and the probe exhibits a strong fluorescent signal. In addition, the probe has good water solubility, high chemical and light stability and excellent mitochondrial targeting capability, and is successfully used for detecting the viscosity change in cell mitochondria before and after drug induction.

In order to achieve the purpose, the invention adopts the following technical scheme:

a mitochondrial viscosity probe having the structural formula:

a preparation method of the mitochondrial viscosity probe comprises the following steps:

(1) dissolving 2, 4-dimethylpyrrole (compound 1) and potassium hydroxide in a solvent under nitrogen, stirring, adding dichloromethane for reaction, cooling the reaction liquid to room temperature, adding water, extracting an aqueous layer, washing the combined organic phase, drying, and separating the crude product by column chromatography to obtain bis (2, 4-dimethyl-1H-pyrrol-1-yl) methane (compound 2);

(2) dissolving the bis (2, 4-dimethyl-1H-pyrrole-1-yl) methane obtained in the step (1) in toluene, then adding triphosgene, refluxing, stirring and reacting, cooling, removing residual phosgene and toluene, and separating a crude product by column chromatography to obtain the carbon dipyrrolone (compound 3);

(3) and (2) mixing bromobenzene and anhydrous tetrahydrofuran, cooling the solution to-78 ℃, adding n-butyllithium solution, mixing and stirring for 30 minutes, adding the anhydrous tetrahydrofuran solution of the pyrrolidione obtained in the step (2), returning the reaction solution to room temperature, stirring overnight, adding hydrochloric acid solution, stirring for 10 minutes, extracting, drying, and separating the crude product by column chromatography to obtain the mitochondrial viscosity probe Cardipy-Vis.

Further, in the step (1), the molar ratio of the 2, 4-dimethylpyrrole to the dichloromethane to the potassium hydroxide is 4:6: 3.

Further, the solvent in the step (1) is dimethyl sulfoxide, the temperature for adding dichloromethane is 40 ℃, and the reaction time is 4 hours.

Further, column chromatography separation developing solvent dichloromethane in the step (1): the volume ratio of the petroleum ether is 1: 4.

further, the molar ratio of bis (2, 4-dimethyl-1H-pyrrol-1-yl) methane to triphosgene in the step (2) is 5: 2.

Further, the temperature of the reflux stirring reaction in the step (2) is 120 ℃ (the boiling point of toluene is 110.6 ℃) and the time is 5 h; the column chromatography separation developing solvent is dichloromethane.

Further, in the step (3), the molar ratio of bromobenzene, n-butyllithium and carbon dipyrrolone is 1:1:0.3, the concentration of n-butyllithium is 1.6mol/L, and the concentration of hydrochloric acid solution is 2 mol/L.

Further, the column chromatography separation developing solvent dichloromethane in the step (3): the volume ratio of methanol was 10: 1.

The application of the mitochondrial viscosity probe in preparing a cell imaging reagent.

The application of the mitochondrial viscosity probe is used for detecting the change of viscosity in cell mitochondria before and after drug induction. The mechanism of the probe for detecting viscosity is shown in fig. 17, in a low-viscosity environment, the C-C bond connecting the fluorophore and the benzene ring can rotate rapidly, the excited state energy of the molecule is consumed by the rapid rotation, the probe returns to the ground state in a non-radiative transition manner, and the fluorescence of the probe is quenched; however, as the viscosity increases, the non-radiative transition process caused by rotation is reduced and the fluorescence of the probe is restored.

Compared with the prior art, the invention has the following advantages:

(1) the probe has good water solubility and strong chemical and light stability;

(2) the probe has natural mitochondrion targeting capability and detects the change of viscosity in cell mitochondrion before and after drug induction;

(3) the probe has good biocompatibility and is expected to be applied to living bodies.

Drawings

FIG. 1 is a drawing of Compound 2¹H NMR chart (CDCl)₃,600MHz)；

FIG. 2 is a drawing of Compound 2¹³C NMR chart (CDCl)₃,150MHz)；

FIG. 3 is a HRMS profile of Compound 2;

FIG. 4 is a drawing of Compound 3¹H NMR chart (CDCl)₃,600MHz)；

FIG. 5 is a drawing of Compound 3¹³C NMR chart (CDCl)₃,150MHz)；

FIG. 6 is a HRMS profile of Compound 3;

FIG. 7 shows the probe Cardipy-Vis¹H NMR Chart (CD)₃CN,600MHz)；

FIG. 8 shows the probe Cardipy-Vis¹³C NMR Chart (CD)₃CN,150MHz)；

FIG. 9 is a HRMS plot of the probe Cardipy-Vis;

FIG. 10 is a graph of the absorbance of the probe Cardipy-Vis in PBS (FIG. 10A) and acetonitrile (FIG. 10B) as a function of concentration (0-40. mu.M).

FIG. 11 is a graph of the normalized fluorescence intensity of the probe Cardipy-Vis and the commercial dyes Cy5.5 and Cy7 under xenon illumination;

FIG. 12 is a graph showing the change of the ultraviolet-visible spectrum and the fluorescence spectrum of a probe Cardipy-Vis in different pH buffer systems with time, wherein Ex is 470nm, slit 5/5nm and PMT 600V, FIG. 12(A) is a graph showing the change of the ultraviolet-visible spectrum and the fluorescence spectrum with time in a buffer system with pH 2-5, FIG. 12(B) is a graph showing the change of the ultraviolet-visible spectrum and the fluorescence spectrum with time in a buffer system with pH 6-9, and FIG. 12(C) is a graph showing the change of the ultraviolet-visible spectrum and the fluorescence spectrum with time in a buffer system with pH 10-12;

FIG. 13(A) is a graph showing the fluorescence spectra of the probe Cardipy-Vis in water and glycerol systems (pH 2-12) at different ratios; FIG. 13(B) is a graph of the fluorescence intensity of the probe at 512nm as a function of viscosity, Ex 470nm, slit 5/10nm, PMT 500V;

FIG. 14 is a co-localized fluorescence image of the probe Cardipy-Vis (1. mu.M)/MitoTracker Red CMXRos (100nM,15min) in Hela cells; the collection wavelength is 490-550nm (λ ex-488 nm)/590-690nm (λ ex-559 nm);

FIG. 15 is a co-localized fluorescence image of the probe Cardipy-Vis (1. mu.M)/LysoTracker Red DND-99(100nM,15min) in Hela cells; the collection wavelength is 490-550nm (λ ex-488 nm)/590-690nm (λ ex-559 nm);

FIG. 16 is an image of probe imaging drug-induced changes in mitochondrial viscosity of HeLa cells; the collection wavelength was 490-550nm (. lamda.ex.488 nm), and FIG. 16(A) shows that the untreated cells did not have any background fluorescence signal; FIG. 16(B) cells treated with probe only exhibited a weak green fluorescence signal; FIG. 16(C) treatment of the cells pre-loaded with nystatin with the probe showed strong green fluorescence signal;

FIG. 17 is a schematic diagram of the mechanism of detecting viscosity of the probe Cardipy-Vis of the invention.

Detailed Description

Example 1

A mitochondrial viscosity probe having the structural formula:

the preparation method of the mitochondrial viscosity probe is characterized by comprising the following steps of:

(1) 2, 4-dimethylpyrrole (0.95g,10mmol) and KOH (0.84g,15mmol) were dissolved in 20mL of dry DMSO under nitrogen, stirred at room temperature for 1h, and CH was added₂Cl₂(0.64g,7.5mmol) and then stirred at 40 ℃ for 4 h. After the reaction solution was cooled to room temperature, 100mL of water was added, the aqueous layer was extracted with diethyl ether, and the combined organic phases were washed with 3X 100mL of water and NaCl, followed by Na₂SO₄And (5) drying. The crude product was separated by column chromatography (dichloromethane/petroleum ether, 1/4) to give bis (2, 4-dimethyl-1H-pyrrol-1-yl) methane (compound 2) as a white solid (0.12g, 11.9%);

¹H NMR(600MHz,CDCl₃)δ6.35(s,2H),5.77(s,2H),5.59(s,2H),2.25(s,6H),2.05(s,6H).¹³C NMR(150MHz,CDCl₃)δ128.1,118.6,117.4,109.6,56.4,145.5,142.6,132.8,132.0,131.7,131.0,127.5,120.1,117.7,115.1,113.0,110.8,99.7,1.96,11.8；ESI-MS[M+H]⁺:calcd for 203.1543,Found 203.1545；

(2) triphosgene (0.178g,0.6mmol) was added with stirring to a solution of bis (2, 4-dimethyl-1H-pyrrol-1-yl) methane (compound 2) (0.3g,1.5mmol) obtained in step (1) in toluene (5mL) and the reaction was stirred at reflux at 120 ℃ for 5H. After cooling, the residual phosgene was removed by nitrogen bubbling. The toluene was removed under reduced pressure and the crude product was isolated by column chromatography (dichloromethane) to afford the carbodipyridone (compound 3) as a white solid (0.118g, 35%);

¹H NMR(600MHz,CDCl₃)δ5.97(s,2H),5.68(s,2H),2.52(s,6H),2.32(s,6H).¹³C NMR(150MHz,CDCl₃)δ169.4,131.2,128.2,124.6,112.7,55.6,12.9,11.9；ESI-MS[M+H]⁺:calcd for 229.1335,Found 229.1333；

(3) 10mL of anhydrous tetrahydrofuran and bromobenzene (157mg, 1.0mmol) were sequentially added to a flask filled with nitrogen, the solution was cooled to-78 ℃, 1.6M n-BuLi (625. mu.L, 1.0mmol) was added thereto, and the mixture was stirred 3 timesAnd 0 minute. A solution of the intermediate carbodipyridone (Compound 3) (76mg, 0.3mmol) from step (2) in dry tetrahydrofuran (10mL) was added slowly to the above solution at the same temperature, and the mixture was allowed to return to room temperature and stirred overnight. Inactivating the reaction by adding 2M hydrochloric acid, stirring for 10min, and adding CH₂Cl₂Extracting, and separating the organic layer with Na₂SO₄Drying and evaporating, and separating the crude product by column Chromatography (CH)₂Cl₂/CH₃OH-10/1) to give probe cardiopy-Vis as an orange solid (0.107g, 95.6%).

¹H NMR(600Hz,CD₃CN)δ7.55(t,J＝7.2Hz,1H),7.47(m,2H),7.28(d,J＝7.2Hz,1H),6.44(s,2H),6.13(s,2H),2.55(s,6H),2.21(s,3H),1.57(s,6H)；¹³CNMR(150MHz,CD₃CN)δ151.9,142.9,142.1,135.2,131.1,130.9,130.3,127.4,127.2,126.1,120.1,58.0,18.2,12.8,12.5；ESI-MS[M]⁺:calcd for 303.1856,Found303.1855.

Example 2

1. Test solution preparation

The probes were made up in 2mM stock with acetonitrile and subsequently diluted to the test concentration with 20mM PBS (pH 7.4) or B-R buffer.

2. Photostability test

The sample solution was placed in a cuvette and continuously irradiated with a xenon lamp (1000w,25A) at room temperature at a distance of 6cm from the cuvette, and the change with time of the fluorescence intensity at the maximum emission point of Cardipy-Vis, Cy5.5 and Cy7 was monitored on a fluorescence spectrometer.

3. Cell culture and fluorescence imaging

HeLa cells were cultured in DMEM (high glucose) medium containing 10% FBS (fetal bovine serum), 100U/mL penicillin G sodium and 100. mu.g/mL streptomycin in a 5% carbon dioxide humidified environment at 37 ℃. Before cell imaging experiments, cells were placed on a 30mm glass-bottomed cell culture dish in advance, left to stand for 12 hours, washed 3 times with Phosphate Buffered Saline (PBS), and then subjected to fluorescence imaging using a Ceiss LMS 710 confocal microscope.

4. Subcellular localization

HeLa cells were cultured in DMEM (high glucose) medium containing 10% FBS (fetal bovine serum), 100U/mL penicillin G sodium and 100. mu.g/mL streptomycin in a 5% carbon dioxide humidified environment at 37 ℃. Before the experiment, the cells were placed on a 30mm glass-bottomed cell culture dish, left to stand for 12 hours, and washed 3 times with Phosphate Buffered Saline (PBS). To confirm that the probe was able to specifically stain mitochondria, HeLa cells were first labeled with LysoTracker Red DND-99(100nM,15min) or MitoTracker Red CMXRos (100nM,15min) under DMEM at 37 ℃ and after washing 3 times with PBS, stained with the probe (1. mu.M, 15min) under DMEM at 37 ℃; after 3 washes with PBS, fluorescence images were taken at the indicated time points. For the probe, the collection wavelength was 500-550nm (λ ex 488 nm); for commercial dyes, the collection wavelength was 590-690nm (λ ex ═ 559 nm).

5. Drug-induced apoptosis assay

The cells were placed on a 30mm glass-bottomed cell culture dish, left to stand for 12 hours, and were treated with nystatin (10. mu.M) for 12 hours before use.

Test results

1. Water solubility and light stability

We first investigated the water solubility of the probe using absorption spectroscopy. As shown in FIG. 10, the absorption wavelengths of the probe Cardipy-Vis in PBS (FIG. 10A) and acetonitrile (FIG. 10B) are 493nm and 494nm, respectively, and show almost the same absorption spectra with substantially unchanged peak-to-shoulder ratio; in addition, titration studies have further shown that the concentration of the probe (0-40. mu.M) is well linear with absorbance, which is sufficient for most bioimaging applications. Furthermore, the probe Cardipy-Vis also has a higher light stability than commercial Cy5.5 and Cy7 when irradiated with Xe lamp for 10min continuously (FIG. 11). The above results show that the cationic property endows the probe Cardipy-Vis with excellent water solubility and light stability, and the two properties are important for biological application.

2. Physical properties of light

In a B-R buffer system, changes of ultraviolet visible absorption spectra and fluorescence spectra of the probe under different pH (2-12) conditions along with time are studied, and as shown in FIG. 12, the probe can stably exist in a pH range of 2-12, which indicates that different pH environments cannot influence the detection of viscosity. The probe itself has a weak fluorescence signal due to the nonradiative transition process caused by the free rotation of the benzene ring, and the change of the fluorescence intensity of the probe in water and glycerol systems with different ratios is tested next in view of the excellent water solubility of the probe, as shown in fig. 13 (a); as shown in FIG. 13(B), the fluorescence intensity of the probe at 512nm gradually increased with an increase in viscosity, and the fluorescence increased by about 12 times.

3. Cell imaging experiments

In order to verify that the probe has natural mitochondrion targeting capability, the cell localization condition of the probe in Hela cells is further tested by using a laser confocal microscope. As shown in fig. 14 and 15, the pearson coefficients were 0.91 and 0.43, respectively, after co-staining the probe with the commercial mitochondrial and lysosomal dyes, respectively, indicating that the probe had a good localization effect with the commercial mitochondrial dyes. The co-localization experiment shows that the dye not only has good cell permeability, but also has a natural mitochondrion targeting function. Researches show that nystatin can induce apoptosis of cells by causing mitochondrial swelling, and in order to verify whether a probe can image mitochondrial viscosity change in the apoptosis process, the cells are stimulated by the nystatin for 0.5 hour in advance and then incubated with the probe; the results show that fig. 16(a) shows that the untreated cells do not have any background fluorescence signal; FIG. 16(B) cells treated with probe only exhibited a weak green fluorescence signal; FIG. 16(C) treatment of the preportioned nystatin cells with the probe showed a strong green fluorescence signal, suggesting that the drug treatment resulted in an increase in mitochondrial viscosity. The above results demonstrate that the probe Cardipy-Vis has natural mitochondrial targeting ability and is successfully used to image mitochondrial viscosity changes during nystatin-induced apoptosis.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A mitochondrial viscosity probe, wherein the probe has the structural formula:

2. a method for preparing the mitochondrial viscosity probe of claim 1, comprising the steps of:

(1) dissolving 2, 4-dimethylpyrrole and potassium hydroxide in a solvent under nitrogen, stirring, adding dichloromethane for reaction, cooling the reaction liquid to room temperature, adding water, extracting an aqueous layer, washing a combined organic phase, drying, and separating a crude product by column chromatography to obtain bis (2, 4-dimethyl-1H-pyrrol-1-yl) methane;

(2) dissolving the bis (2, 4-dimethyl-1H-pyrrole-1-yl) methane obtained in the step (1) in toluene, then adding triphosgene, refluxing, stirring and reacting, cooling, removing residual phosgene and toluene, and separating a crude product by column chromatography to obtain the carbon dipyrrolone;

(3) and (3) mixing bromobenzene and anhydrous tetrahydrofuran, cooling the solution to-78 ℃, adding n-butyllithium solution, mixing and stirring for 30 minutes, adding the anhydrous tetrahydrofuran solution of the pyrrolidione obtained in the step (2), returning the reaction solution to room temperature, stirring overnight, adding hydrochloric acid solution, stirring for 10 minutes, extracting, drying, and separating the crude product by column chromatography to obtain the mitochondrial viscosity probe.

3. The method for preparing a mitochondrial viscosity probe according to claim 2, wherein the molar ratio of 2, 4-dimethylpyrrole, dichloromethane and potassium hydroxide in the step (1) is 4:6: 3.

4. The method for preparing a mitochondrial viscosity probe according to claim 2, wherein the solvent in step (1) is dimethyl sulfoxide, the temperature for adding dichloromethane is 40 ℃, and the reaction time is 4 hours; the column chromatography developing solvent dichloromethane: the volume ratio of the petroleum ether is 1: 4.

5. the method of claim 2, wherein the molar ratio of bis (2, 4-dimethyl-1H-pyrrol-1-yl) methane to triphosgene in step (2) is 5: 2.

6. The method for preparing a mitochondrial viscosity probe according to claim 2, wherein the temperature of the reflux stirring reaction in the step (2) is 120 ℃ and the time is 5 h; the column chromatography separation developing solvent is dichloromethane.

7. The method for preparing a mitochondrial viscosity probe according to claim 2, wherein the molar ratio of bromobenzene, n-butyllithium and pyrrolidione in the step (3) is 1:1:0.3, the concentration of the n-butyllithium solution is 1.6mol/L, and the concentration of the hydrochloric acid solution is 2 mol/L.

8. The method for preparing a mitochondrial viscosity probe according to claim 2, wherein the column chromatography separation developing solvent dichloromethane in the step (3): the volume ratio of methanol was 10: 1.

9. Use of the mitochondrial viscosity probe of claim 1 for the preparation of a reagent for cell imaging.

10. Use of the mitochondrial viscosity probe of claim 1 for the preparation of a reagent for detecting changes in viscosity in the mitochondria of a cell before and after drug induction.